scholarly journals Assessment of Hypercoagulable State in Whole Blood in Sepsis Patients Using a Novel Microfluidic Dielectric Sensor

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1882-1882
Author(s):  
Sina Pourang ◽  
Michael A Suster ◽  
Pedram Mohseni ◽  
Lalitha V Nayak
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Stock Options ◽  
Research Funding ◽  
Hypercoagulable State ◽  
Blood Samples ◽  
Healthy Donors ◽  
Dielectric Sensor ◽  
Peak Parameter ◽  
Coagulation Status

Abstract Background: There is an intimate link between inflammation and thrombosis, and patients with pro-inflammatory/infectious disorders develop a hypercoagulable state. Extant coagulation assays are unable to distinguish the pro-coagulant state of a patient's blood, require 2-3 mL of blood, and take 2-3 hours for processing. These assays are also typically examined in plasma and do not represent the contribution of blood cellular elements that participate in thrombosis in vivo. Thus, a point-of-care device for rapid, comprehensive assessment of whole blood coagulation is crucial to ensure appropriate and timely evaluation in critically ill patients. We have introduced a microfluidic sensor (ClotChip) that uses dielectric spectroscopy to provide such an assessment in a handheld platform. We have shown in clinical studies in patients with a hypocoagulable state that ClotChip is sensitive to both coagulation factor and platelet defects, allowing for a global assessment of blood coagulation status using <10 µL of whole blood and in <30 min. In this study, we optimized ClotChip to assess the blood coagulation status in patients with a hypercoagulable state. Methods: Citrated blood samples from 12 patients with a diagnosis of sepsis and 11 healthy donors as controls were obtained under an IRB-approved protocol and tested with ClotChip within 2 hours of collection. ClotChip readout curve was calculated as the temporal variation of blood dielectric permittivity at 1 MHz, and the time to reach a permittivity peak (T peak) was taken as an indicator of coagulation time based on our prior studies. To increase the sensitivity of the ClotChip T peak parameter to a hypercoagulable state, we used two different anticoagulants, recombinant thrombomodulin (rTM) and activated protein C (APC). To optimize the anticoagulant concentration, whole blood samples from healthy donors were treated in vitro with lipopolysaccharide to mimic a pro-coagulant state of blood and tested with ClotChip after adding various concentrations of rTM and APC. We concluded that a concentration of 5 µg/mL for rTM and 10 µg/mL for APC would result in an optimal change in T peak for detecting the pro-coagulant state. Since heparin (or lovenox) is routinely used in hospitalized patients, sepsis and control samples were pretreated with hepzyme at a final concentration of 2 IU/mL to reverse the heparin effect. The T peak parameter was measured and compared in (i) hepzyme only-, (ii) rTM-, and (iii) APC-treated samples. Data are reported as mean ± standard deviation. Two-tailed t test is used to test for statistical significance between groups, and P < 0.05 is considered statistically significant. In box-and-whiskers plots, the box represents the range from the first to the third quartile, the horizontal line represents the median, plus sign (+) represents mean of the data; whiskers extend to the maximum and minimum data values, and dots represent individual subject data. Results: In hepzyme only-treated samples, T peak was significantly prolonged at 478±137 sec in sepsis samples, as compared to 357±58 sec in controls (Figs. 1A, 1B). rTM treatment resulted in T peak of 503±128 sec for sepsis samples and 443±81 sec for controls, whereas APC treatment resulted in T peak of 1,095±850 sec for sepsis samples and 477±71 sec for controls (Figs. 1A, 1B). Although T peak was prolonged at baseline in hepzyme only-treated sepsis samples, no further prolongation was noted with rTM treatment (difference in T peak of 24±94 sec; Fig. 1C), as compared to rTM-treated controls (difference in T peak of 85±40 sec; Fig. 1C). However, with a difference in T peak of 616±804 sec, the APC-treated sepsis samples exhibited T peak prolongation when compared to hepzyme only-treated sepsis samples, whereas the APC-treated controls did not (difference in T peak of 119±64 sec; Fig. 1D). A comparison between the APC- and rTM-treated samples revealed a significant prolongation of T peak in sepsis samples (difference in T peak of 591±815 sec) when compared to controls (difference in T peak of 30±66 sec; Fig. 1E). Conclusions: Our studies identify a unique coagulation profile in sepsis patient blood using a microfluidic dielectric sensor. These data suggest that the addition of rTM or APC can enhance the sensitivity of the ClotChip T peak parameter for detecting the pro-coagulant state in whole blood. Ongoing studies are examining the coagulation profile in other pro-inflammatory and infectious states. Figure 1 Figure 1. Disclosures Suster: XaTek Inc.: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Mohseni: XaTek Inc.: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Nayak: BioChip Labs: Current Employment.

scholarly journals Characterization of Two Anti-P-Selectin Monoclonal Antibodies (mAbs): Crizanlizumab Shows Comparable or Stronger Effects Versus Inclacumab across Cell Adhesion Assays in Vitro

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2032-2032
Author(s):  
Tina Rubic-Schneider ◽  
Prithu Sundd ◽  
David Ledieu ◽  
Déborah Garcia ◽  
Jeannine Hehlen ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Healthy Volunteers ◽  
Binding Affinity ◽  
Whole Blood ◽  
Stock Options ◽  
Research Funding ◽  
Blood Samples ◽  
Selectin Binding ◽  
Privately Held

Abstract Background: Vaso-occlusive crises (VOCs) are the hallmark of sickle cell disease (SCD) and are associated with significant morbidity and mortality. Crizanlizumab, a first-in-class humanized anti-P-selectin IgG2 mAb, is approved in >40 countries to reduce/prevent VOCs in SCD patients aged ≥16 yrs. Inclacumab, a fully human anti-P-selectin IgG4 mAb, is in clinical development for SCD. In vitro data suggest that inclacumab may show stronger affinity to P-selectin and greater maximal inhibition of cell-cell interactions vs crizanlizumab (Geng et al ASH 2020). Aim: We investigated in blinded experiments whether crizanlizumab and inclacumab can be differentiated in terms of P-selectin binding and inhibition of P-selectin-mediated cell-cell interactions, via 5 different in vitro assays, including testing of blood samples from healthy volunteers and SCD patients. Methods: For both mAbs, we assessed: P-selectin affinity (surface plasmon resonance [SPR]/Biacore assay); inhibition of adhesion of P-selectin-expressing cells to P-selectin glycoprotein ligand-1 (PSGL-1; cell adhesion bioassay); inhibition of platelet aggregation in native blood from healthy volunteers (whole blood impedance aggregometry [WBA] and flow cytometry-based platelet-leukocyte aggregate [PLA] assays); and inhibition of adhesion of SCD patient whole blood or isolated leukocytes to P-selectin-coated substrate under physiologically relevant shear rates in microfluidic systems (flow adhesion bioassays). Results: The SPR/Biacore assay indicated that both mAbs recognize P-selectin with binding affinities of the same order of magnitude, although there was a trend towards higher binding affinity with inclacumab (mean equilibrium dissociation constant [K D ± standard deviation]: 12.4 ± 1.0 nM [crizanlizumab] vs 6.7 ± 0.7 nM [inclacumab]). However, the function-related cell adhesion bioassay revealed a significant difference between the mAbs in their ability to inhibit adhesion of P-selectin-expressing cells to PSGL-1. Crizanlizumab showed stronger inhibition than inclacumab (potency vs crizanlizumab reference standard: 98.0% for the crizanlizumab control sample [95% CI 85.2‒112.7%] and 26.2% for the inclacumab sample [95% CI 20.7%‒32.6%]). Native whole blood samples for WBA and PLA assays were provided by 12 and 10 healthy volunteers, respectively. Blocking of platelet (WBA assay) or platelet-leukocyte aggregation (PLA assay) in whole blood was comparable for crizanlizumab and inclacumab, with a clear trend towards stronger inhibition by crizanlizumab. In the WBA assay, the half maximal inhibitory concentration (IC 50) was twice as high with inclacumab (10.36 μg/mL) as crizanlizumab (5.11 μg/mL); similar data were seen in the PLA assay (Figure 1; IC 50: 4.11 vs 2.81 μg/mL for inclacumab vs crizanlizumab). Blood samples were provided by 9 SCD patients for the flow adhesion bioassays. Dose-dependent inhibition of adhesion of whole blood and isolated leukocytes to P-selectin was seen with both crizanlizumab and inclacumab, with no difference observed between them (Figure 2). A mAb concentration of 10 μg/mL resulted in statistically significant inhibition of adhesion by 60‒75%; further increasing the mAb concentration did not result in stronger inhibition of adhesion beyond this threshold. In relation to the clinically approved crizanlizumab dose (5.0 mg/kg), a concentration of 100 μg/mL corresponds with the maximum plasma concentration (C max) and ~10-15 μg/mL corresponds with the trough concentration (C trough). Conclusions: While the SPR/Biacore data suggested higher P-selectin binding affinity for inclacumab compared with crizanlizumab, there was a stronger inhibition of P-selectin-mediated cell adhesion with crizanlizumab vs inclacumab in other well-characterized functional in vitro assays. In microfluidic flow adhesion bioassays, blockage of cell adhesion from SCD whole blood and leukocytes was comparable for both mAbs. In summary, these data indicate that comparable or stronger blockage of cell adhesion with crizanlizumab vs inclacumab does not require superior P-selectin binding affinity. The data from healthy volunteers will be complemented by data from SCD patients for the WBA and PLA assays. Ultimately, clinical data are required to evaluate potential differences in the profiles and efficacy of crizanlizumab and inclacumab as treatments for SCD patients. Figure 1 Figure 1. Disclosures Rubic-Schneider: Novartis Pharma AG: Current Employment. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ledieu: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Garcia: Novartis Pharma AG: Current Employment. Hehlen: Novartis Pharma AG: Current Employment. Burnet-Merlin: Light Chain Bioscience - Novimmune SA: Current Employment; Novartis Pharma AG: Ended employment in the past 24 months. Cochin de Billy: Novartis Pharma AG: Current Employment. Greutmann: Novartis Pharma AG: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Krӧner: Novartis Pharma AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: WO2021087050A1. Verneret: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Bruederle: Novartis Pharma AG: Current Employment. Gao: Functional Fluidics: Current Employment. Dajee: Novartis: Current Employment.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1845-1845
Author(s):  
Suzanne Trudel ◽  
Susan Lee ◽  
Christopher J. Kirk ◽  
Nashat Gabrail ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Whole Blood ◽  
Research Funding ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Advisory Committees ◽  
Post Dose ◽  
Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

scholarly journals Assessment of the Effect of Crizanlizumab on Red Blood Cell Adhesion to Endothelial Cells Using a Standardized Endothelium-on-a-Chip Microfluidic Platform

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2021-2021
Author(s):  
Erdem Kucukal ◽  
Aaron Wolfe ◽  
Ryan Kocevar ◽  
Lalitha V Nayak ◽  
Andreas Bruederle ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Microfluidic Channels ◽  
Blood Samples ◽  
Privately Held

Abstract Background: Chronic upregulation of P-selectin (P-sel) on blood cells and the endothelium leads to abnormal red blood cell (RBC) adhesion to endothelial cells, significantly contributing to vaso-occlusive crises (VOCs), which are a major cause of morbidity and mortality in patients with sickle cell disease (SCD). Crizanlizumab (criz, a.k.a. SEG101) is a humanized anti-P-sel monoclonal antibody and has recently been approved by the Food and Drug Administration to reduce the frequency of VOCs in SCD patients. Here, we report in vitro assessment of the effect of criz on patient-specific RBC adhesion to heme-activated human endothelial cells using a standardized endothelialized microfluidic platform, the Endothelium-on-a-chip. Methods: Whole blood samples were collected from 13 subjects with SCD (13 HbSS and 1 HbSC) in EDTA vacutainers. RBCs were isolated via centrifugation from whole blood and then resuspended in basal cell culture medium (EBM, Lonza, Morristown, USA) at a hematocrit of 20% buffered with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured within the microfluidic channels at 15 dyne/cm 2 for at least 48 hours prior to experiments. For long-term activation, HUVECs were treated with 40 µM heme for 4 hours +/- 100 µg/ml criz for 1 hour followed by injection of blood samples through the microfluidic channels. For short-term activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml criz and injected through the microfluidic channels for 15 minutes. Thereafter, non-adherent RBCs were rinsed via either only heme-containing EGM or heme- and criz-containing EGM, and the remaining RBCs were quantified based on published methods [1]. Student's t-test was used to calculate statistical significance. Results: We found that 4-hour heme activation of HUVECs resulted in significantly elevated RBC adhesion compared to baseline although adhesion levels were heterogenous among the patient population (Fig. 1A, 1671±522 vs 17±4, p<0.05). Treatment of 4-hour heme-activated HUVECs with criz did not significantly decrease RBC adhesion (Fig. 1A, 1170±413 vs 1671±522, p>0.05), while we observed lower RBC adhesion to criz treated HUVECs for certain subjects (Fig. 1B). By contrast, criz treatment significantly reduced the number of adherent RBCs to 15-min heme-activated HUVECs (Fig. 1C, 135±40 vs 1513±617, p<0.05). Next, we assessed whether criz would disrupt already established adhesive interactions between RBCs and 15-min heme-activated HUVECs. To do so, we first allowed RBCs to adhere to heme-activated HUVECs (for 15-min) and then rinsed the microchannels (at 10 μl/min) via either a heme- or both heme- and criz-containing solution (for 15 min). We then quantified the number of adherent RBCs at min=0 and min=15. While only 10% of the adherent RBCs remained in the microchannels following a 15-minute wash with criz, this ratio was 60% without criz (Fig. 1E). Discussion: Our results show that the magnitude of inhibition of RBC adhesion to HUVECs with criz correlated with the duration of heme-activation (4 hours vs 15 minutes). This is likely due to variable levels of different adhesion molecules on acute or chronically activated HUVECs. For instance, it has been shown that P-selectin is rapidly translocated to the cell surface following heme activation [1], but its concentration on cell surface significantly decays with time. Previous experiments have shown that sickle RBCs can adhere to cell adhesion molecules such as ICAM-1 [2], which mechanistically may play a role in the case of a chronically activated endothelium. We are currently exploring whether criz would also reduce RBC adhesion to acutely activated endothelial cells that are under chronic stress. These preliminary results suggest that the Endothelium-on-a-chip, as partner in novel therapeutic studies, could help monitoring dynamics of targeted therapies in SCD patients during drug development and in clinical trials. Acknowledgements: This work was funded by Novartis. The authors would like to thank the Ohio Third Frontier Technology Validation and Start-up Fund (TVSF) and National Science Foundation Phase-I Small Business Technology Transfer (STTR) award, which supported this work in part. Crizanlizumab was donated by Novartis. References: 1. Kucukal, E., et al., American Journal of Hematology, 2018. 93(8): p.1050-60 2. Kucukal, E., et al., Blood Advances, 2020. 4(15):3688-98 Figure 1 Figure 1. Disclosures Kucukal: BioChip Labs: Current Employment, Patents & Royalties. Kocevar: BioChip Labs: Current Employment. Nayak: BioChip Labs: Current Employment. Bruederle: Novartis Pharma AG: Current Employment. Zak: XaTek: Current Employment, Current holder of stock options in a privately-held company; BioChip Labs: Current Employment, Current holder of stock options in a privately-held company; TecTraum Inc: Current Employment, Current holder of stock options in a privately-held company. Gurkan: Dx Now Inc.: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Biochip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties.

scholarly journals Antibody and T-Cell Responses to COVID-19 Vaccination in Myeloproliferative Neoplasm Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 316-316
Author(s):  
Joan How ◽  
Kathleen M.E. Gallagher ◽  
Yiwen Liu ◽  
Ashley DeMato ◽  
Katelin Katsis ◽  
...  
Keyword(s):  
T Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Research Funding ◽  
T Cell Responses ◽  
Spike Protein ◽  
Healthy Controls ◽  
Whole Blood Assay ◽  
Post Vaccination ◽  
Cell Responses

Abstract The efficacy of COVID-19 vaccines in cancer populations remain unknown. Myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) remain a vulnerable patient population and are immunocompromised due to impaired innate and adaptive immunity, heightened inflammation, and effects of ongoing treatment. We evaluate antibody and T-cell responses in MPN patients following completion of the BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) COVID-19 vaccine series. Patients with a known diagnosis of MPN presenting at Massachusetts General Hospital and eligible for COVID-19 vaccination were recruited. All participants gave informed consent and the study protocol was approved by the Institutional Review Board. 33 MPN patients were enrolled and 23 patients completed vaccination. Baseline and post-vaccination peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) isolated. 26 vaccinated participants with no history of malignancy were included as healthy controls (PMID 33972942). Baseline characteristics are tabled below. Qualitative ELISA for human IgG/A/M against SARS-CoV-2 spike protein using donor serum was performed per manufacturer instructions. Seroconversion occurred in 22/23 (96%) of MPN patients and 25/26 (96%) of healthy controls (Figure). To measure SARS-CoV-2 T-cell immunity, an IFNγ ELISpot assay previously developed in convalescent and vaccinated healthy individuals was used. Freshly isolated PBMCs from patients were stimulated with commercially available overlapping 15mer peptide pools spanning the SARS-CoV-2 spike and nucleocapsid proteins. Given its size, the spike protein was split into two pools (Spike A or B). IFNγ-producing T-cells were quantified by counting the median spot forming units (SFU) per 2.5x10 5 PBMCs from duplicate wells. A positive threshold was defined as >6 SFUs per 2.5x10 5 PBMCs to either Spike A or B after subtraction of background, based on prior receiver operator curve (ROC) analysis of ELISpot responses (sensitivity 90% specificity 92%). Post-vaccination ELISpot responses occurred in 21/23 (91%) of MPN patients and 26/26 (100%) of healthy controls (p=0.99) (Figure). The median SFU to total spike protein (Spike A+B) increased after vaccination in both MPN patients (0 to 38, p=0.02) and healthy controls (6 to 134, p=0.002). MPN patients had significantly lower median SFU's on post-vaccination ELISpot compared to healthy controls (38 vs 134, p=0.044), although this was not significant after adjusting for age in multivariable logistic regression. MF patients had the lowest seroconversion and ELISpot response rates, and lowest median post-vaccination SFUs, although this was not significant. There were no other differences in post-vaccination SFUs with regards to gender, vaccine type, number of days post-vaccine, treatment, and absolute lymphocyte count. Whole-blood assay based on the in vitro diagnostic QuantiFERON TB Gold Plus assay was also used to assess T-cell response. Heparinized whole blood from donors was stimulated with S1 and S2 subdomains for the SARS-CoV-2 spike protein, with measurement of IFNγ released into plasma with the QuantiFERON ELISA. IFNγ release of >0.3 IU/mL was considered a positive threshold, based on prior ROC analysis (sensitivity and specificity 100%). MPN patients had significantly lower IFNγ response rates compared to healthy controls (57% versus 100%, p=0.003) (Figure). Our findings demonstrate robust antibody and T-cell responses to BNT162b2 and mRNA-1273 vaccination in MPN patients, with >90% serologic and ELISpot responder rates. We detected subtle differences in T-cell responses in MPN patients compared to healthy controls. MPN patients had lower median post-vaccination ELISpot SFUs and lower rates of T-cell responses on IFNγ-whole blood assay compared to healthy controls. As the whole blood assay uses whole protein antigen rather than peptide pools, differences from ELISpot testing may reflect deficiencies in antigen processing and presentation. It is unclear whether these subtle differences translate into less clinical protection from COVID-19, or to what extent our results are confounded by the older age of MPN patients. Further evaluation of B and T-cell responses to COVID-19 vaccination in a larger sample size of MPN patients is warranted. Figure 1 Figure 1. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. Maus: Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; AstraZeneca: Consultancy; Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company. Hobbs: AbbVie.: Consultancy; Incyte Corporation: Research Funding; Novartis: Consultancy; Bayer: Research Funding; Merck: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Celgene/Bristol Myers Squibb: Consultancy.

scholarly journals Differential Neutralization of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban By Andexanet Alfa As Measured By Whole Blood Thromboelastographic Analysis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1155-1155
Author(s):  
Siddharth Mehrotra ◽  
Debra Hoppensteadt ◽  
Walter Jeske ◽  
Omer Iqbal ◽  
Alfonso J Tafur ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Surrogate Marker ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Saline Control ◽  
Unequal Variances ◽  
Healthy Donors ◽  
Andexanet Alfa ◽  
The Individual

Introduction/Background: Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed as an antidote to neutralize the bleeding effects of oral FXa inhibitors such as, Apixaban and Rivaroxaban. The relative biological effect of these drugs have been investigated using various clot based and amidolytic methods for FXa inhibition. This Factor Xa inhibitory activity acts as a surrogate marker for the circulating level of these agents. We have recently reported that the FXa activity of these Anti-Xa agents does not fully reflect their biologic spectrum (JCath 25,1-11,2019). Whole blood assays such as thromboelastographic analysis represent a global assay which takes into account both the plasmatic and cellular components of blood and provides a more physiologic endpoint to study the anticoagulant effects of these drugs. The purpose of this study was to investigate the anticoagulant effects of currently available oral Anti-Xa agents such as Apixaban, Betrixaban, Edoxaban, and Rivaroxaban and their relative neutralization by AA in terms of such thromboelastographic parameters as R, K, Angle and MA. Materials and Methods: Analysis was carried out in whole blood using thromboelastography (TEG) using the TEG 5000 Hemostasis System (Haemonetics Corp, Massachusetts). Blood was drawn from healthy donors in individual groups (n=5-10) into 3.2% citrated tubes. In the TEG cup for testing, 0.2 M CaCl2, saline (with a filler and control), each of the individual FXa Inhibitors at a final concentration(FC) of 1 ug/mL, AA at FC of both 100 ug/mL and 50 ug/mL were tested for the relative neutralization of the anticoagulant effects. TEG parameters such as R-time, K-time, angle and MA were measured. All results were compiled individually for the saline control, 100 ug/mL AA and 50 ug/mL AA supplemented systems. Statistical analysis was carried out via an F test for equality of variances followed by the appropriate t test for equal or unequal variances. Results: When comparing the anticoagulants directly to one another, it was observed that Edoxaban shows the strongest anticoagulant effects in both R and K time followed by Betrixaban, then Rivaroxaban which were very similar in their anticoagulative effects, with Apixaban showing the weakest anticoagulant effect as shown in Table 1A. In the reversal studies as shown in Table 1B, as measured by various TEG parameters, R-Time, AA (FC=100 ug/mL) showed full neutralization effects in Apixaban (p=.027), Betrixaban(p=<.01), Edoxaban(p<.01), and Rivaroxaban(p<.01). In K-time, Betrixaban and Edoxaban were fully neutralized (respectfully p=.049 and p=.035) with partial neutralizations of Apixaban and Rivaroxaban. No significant neutralization was noted in the Angle and MA. AA at 50 ug/mL showed full neutralizations, in R-time, in Betrixaban and Rivaroxaban (Betrixaban[p<.01] and Rivaroxaban[p=.0287]), AA at this concentration showed partial neutralizations of Apixaban and Edoxaban. In K-time, AA showed full neutralization of Betrixaban and Edoxaban (Betrixaban[p=<.01] and Edoxaban[p<.01]). Apixaban and Rivaroxaban saw no neutralization effect by AA at FC=50 ug/mL in K-time. AA did not exhibit any significant neutralization effects in the Angle or MA parameters. Summary and Conclusion: All of the 4 Agents produced measurable anticoagulative activities at 1 ug/mL as measured by the TEG parameters. Edoxaban exhibited the strongest anticoagulative effect followed by Betrixaban and Rivaroxaban whereas Apixaban showed much weaker anticoagulant effects. AA FC=100 ug/mL showed much stronger, consistent, and complete neutralization effects of all of the 4 FXa Inhibitors when compared to AA at FC=50 ug/mL. These results strongly suggest that regardless of the variable anticoagulative effect exhibited by the FXa Inhibitors, AA at FC=100ug/mL fully neutralized the anticoagulant effects of this agent as measured by the TEG parameters. AA is shown to be the most effective in neutralizing Betrixaban in R-Time and K-Time at both concentrations of AA. AA was seen to neutralize Apixaban the least. It can be concluded that the effect of AA as a neutralizing agent is both drug and donor dependent and therefore dosage adjustment may be needed for the optimal clinical outcome with this antidote. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

scholarly journals Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4662-4662
Author(s):  
Debnath Maji ◽  
Michael A Suster ◽  
Divyaswathi Citla Sridhar ◽  
Maria Alejandra Pereda ◽  
Janet Martin ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Point Of Care ◽  
Blood Samples ◽  
Healthy Group ◽  
Significant Difference ◽  
Bleeding Score ◽  
Bleeding History

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and > 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P < 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score >12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score <12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P < 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P < 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P < 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (<10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents & Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents & Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents & Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents & Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

Monitoring Fibrin Polymerization Effects on Whole Blood Coagulation Using a Microfluidic Dielectric Sensor

2020 ◽  
Author(s):  
Sina Pourang ◽  
Debnath Maji ◽  
Ujjal D. S. Sekhon ◽  
Anirban Sen Gupta ◽  
Michael A. Suster ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Fibrin Polymerization ◽  
Dielectric Sensor

scholarly journals Natalizumab Blocks VLA-4 Mediated Red Blood Cell Adhesion and Is a Potential Therapy for Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 221-221 ◽  
Author(s):  
Patrick C. Hines ◽  
Sriram Krishnamoorthy ◽  
Jennell White ◽  
Dipti Gupta ◽  
Arjan van der Flier ◽  
...  
Keyword(s):  
Whole Blood ◽  
Blood Cells ◽  
Research Funding ◽  
Healthy Donor ◽  
Cell Disease ◽  
Flow Conditions ◽  
Employment Equity ◽  
Healthy Donors ◽  
Equity Ownership ◽  
Adhesive Interactions

Abstract Sickle cell disease (SCD) is caused by a point mutation in the beta-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive events (VOCs) and chronic hemolytic anemia. Among other abnormalities, sickle red blood cells (RBCs) are more adhesive than normal RBCs. Sickle RBC adhesion is an important pathway leading to both VOC and hemolysis: adhesive interactions promote the formation of blood flow-obstructing heterocellular aggregates that induce ischemic tissue damage and slow the transit of RBCs through the vasculature, promoting sickle hemoglobin polymerization and hemolysis. The capacity of sickle RBCs to adhere to endothelium positively correlates with disease severity. Interventions that reduce sickle RBC adhesion, in particular that of reticulocytes, by blocking specific molecular targets may limit or prevent disease sequelae. Very Late Antigen – 4 (VLA-4 or α4β1 integrin) is an important integrin on reticulocytes that mediates adhesive interactions to endothelial and plasma vascular cell adhesion molecule - 1 (VCAM-1), plasma fibrinogen, and other ligands. VLA-4 is expressed on the surface of sickle reticulocytes, with levels decreasing during maturation such that mature RBCs do not express surface VLA-4. Natalizumab is a recombinant humanized antibody that binds to the α4 subunit of VLA-4 and is used to treat multiple sclerosis (MS) and Crohn’s Disease (CD) by preventing leukocyte trafficking into tissues at sites of inflammation. We investigated the ability of natalizumab to block VLA-4 in the context of sickle whole blood, and evaluated the effect on sickle reticulocyte, mature erythrocyte, and leukocyte adhesion during physiologic flow conditions. Whole blood samples obtained from SCD donors were analyzed for saturation binding of natalizumab to surface VLA-4 on leukocytes and reticulocytes using flow cytometry. Up to 20% of reticulocytes from SCD donors (n=13) were positive for VLA-4 surface staining, whereas VLA-4 was undetectable on the surface of reticulocytes from healthy donors (n=4). VLA-4 on SCD reticulocytes and mononuclear leukocytes was saturated by natalizumab concentrations lower than known plasma trough concentrations achieved in MS and CD patients after natalizumab treatment, with SCD reticulocyte EC50 = 0.11 ± 0.01 μg/mL and SCD leukocyte EC50 = 0.16 ± 0.01 μg/mL (n=6). This translates to a binding affinity of 0.7 to 1.3 nM, similar to that found for healthy donor leukocytes. Both whole blood cells and isolated leukocytes from SCD donors adhered to immobilized VCAM-1 during physiologic flow conditions simulating post-capillary venules (shear stress =1 dynes/cm2) using a microfluidic flow-based adhesion system. Leukocytes from SCD donors adhered more to VCAM-1 (Mean = 20.7 + 10.0 from n=7) than leukocytes from healthy donors (Mean = 5.0 + 1.4 from n=5). The adhesion of leukocytes and reticulocytes to VCAM-1 was blocked by natalizumab in a dose-dependent manner and as a function of natalizumab saturation of cell surface VLA-4. Therapeutic IgG4 antibodies, including natalizumab, undergo chain shuffling in vivo with endogenous IgG4, leading to mono-specific IgG4 molecules, with one Fab arm specific to the antigen it was raised against. Compared to divalent natalizumab, monovalent chain-shuffled natalizumab bound to SCD reticulocytes at a7-fold higher EC50 and inhibited SCD reticulocyte adhesion to VCAM-1 at higher antibody concentrations (10 and 1 µg/mL). While increased, these values are still below trough natalizumab levels achieved in natalizumab-treated patients. In summary, natalizumab bound VLA-4 on the surface of SCD reticulocytes and leukocytes similarly to healthy donor leukocytes and blocked adhesion of SCD reticulocytes and leukocytes to immobilized VCAM-1 under shear conditions. Natalizumab binding and adhesion inhibition occurred at plasma concentrations similar to those seen in natalizumab treated MS and CD patients. Based on these findings, natalizumab may have potential as an anti-adhesive therapy for SCD. Further clinical studies are needed to evaluate the safety and efficacy of natalizumab in SCD. Disclosures Hines: Biogen Idec: Research Funding. Krishnamoorthy:Biogen Idec: Employment, Equity Ownership. White:Biogen Idec: Research Funding. Gupta:Biogen Idec: Employment, Equity Ownership. van der Flier:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership. Hobbs:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership.

scholarly journals Continuous Thrombin Generation in Whole Blood: New Applications

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4979-4979
Author(s):  
Shannon M Prior ◽  
Kenneth G Mann ◽  
Kalev Freeman ◽  
Saulius Butenas
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Blood Cells ◽  
Thrombin Generation ◽  
Sodium Citrate ◽  
Lag Phase ◽  
Packed Red Blood Cells ◽  
Healthy Donors ◽  
Hemostatic Tests ◽  
Peak Value

Abstract Background. Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation. Methods. Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay. Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids. Results. Whole Blood A. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower. B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase. C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF. a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold. b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold. c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG. d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin. D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa. Packed Red Blood Cells (pRBC) In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile. Conclusions. This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders. Disclosures Mann: Haematologic Technologies, Inc.: Employment, Equity Ownership; Baxter: Consultancy; Bayer: Consultancy; Biogen IDEC: Consultancy; CSL Behring: Consultancy; Merck: Consultancy; Pfizer: Consultancy; The Medicines Company: Consultancy; XO1: Consultancy; Vascular Solutions: Consultancy; Stago: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document